One-pot synthesis of tetrahydropyrimidinecarboxamides enabling in vitro anticancer activity: a combinative study with clinically relevant brain-penetrant drugs

In this study, we describe a one-pot three-component synthesis of bioactive tetrahydopyrimidinecarboxamide derivatives employing lanthanum triflate as a catalyst. Out of the synthesized compounds, 4f had the most potent anti-cancer activity and impeded cell cycle progression effectively. Anti-cancer bioactivity was observed in 4f against liver, breast, and lung cancers as well as primary patient-derived glioblastoma cell lines. Compound 4f effectively inhibited the 3D neurosphere formation in primary patient-derived glioma stem cells. Specifically, 4f exhibited synergistic cytotoxicity with the EGFR inhibitor that is the clinical epidermal growth factor receptor inhibitor osimertinib. 4f does not exhibit anti-kinase activity and is cytostatic in nature, and further work is needed to understand the true molecular target of 4f and its derivatives. Through our current work, we establish a promising tetrahydopyrimidinecarboxamide-based lead compound with anti-cancer activity, which may exhibit potent anti-cancer activity in combination with specific clinically relevant small molecule kinase inhibitors.


Introduction
Dihydropyrimidinones (DHPMs) produced via the Biginelli reaction have received signicant attention owing to their diverse biological applications, such as acetylcholinesterase inhibitors, 1 HIV-1 replication inhibitors, 2 urease inhibitors, 3 calcium channel blockers, 4 antimicrobial agents, 5 antioxidant agents, 6 nonsteroidal RORa agonists, 7 anti-cancer cytotoxic agents, 8 and antitubercular agents. 9From this DHPM family of compounds, monastrol emerged as a key lead compound. 10,11onastrol has signicant anticancer properties (Fig. 1) and prevents metastasis by impeding the movement of kinesin spindle protein Eg5, 12 which associates with microtubules of the mitotic spindle 13 and engages in intracellular transport and cell division. 14Dihydropyrimidine derivatives allosterically inhibit Eg5-mediated microtubule organisation and drive the antimetastatic activities. 15Monastrol was rst observed to cause cell cycle disruption in Xenopus models 16 and later monastrol and its derivatives were reported to have robust anti-proliferative activities against cancer cells. 17Multiple research groups have since developed several DHPMs that exhibited antiproliferative properties against other human solid tumour cell lines (Fig. 1).
In the current study, we wanted to utilise our long-standing successful strategy 11,[18][19][20][21][22][23][24][25] of utilising the Biginelli reaction to synthesize benzyloxy derivatives of DHPM to obtain new bioactive, anti-cancer compounds.][31][32] Similarly, the incorporation of a 1,2,3-triazole moiety into various compounds has shown signicant pharmacological potential, such as anticancer, [33][34][35][36][37][38] antimicrobial, 33,39 anti-inammatory, 40 antitubercular, 41 anti-HIV, 42 and antiviral effects. 33][45][46][47][48] The aim of this study was to utilise the Biginelli reaction to integrate the 1,4-disubstituted 1,2,3-triazole and benzyloxyphenyl group using a wide variety of catalysts like Lewis acids, salts, or ionic liquids to generate phenyloxymethyl-1,2,3-triazole derivatives with potentially distinct anticancer attributes.Furthermore, we employed lanthanum triate as a catalyst that acts as a Lewis acid resulting in excellent yield over a short reaction period.From the series of compounds synthesized, we identied one derivative to have potent anti-proliferative effects across multiple solid tumour cell lines and effectively impede the cell cycle with a reduction in cancer stem cell numbers in primary patient-derived cancer cells.The derivative also induced cytotoxicity in combination with FDA-approved clinical drugs thus suggesting a novel paradigm in our efforts to utilise the Biginelli reaction and green catalysts to generate anti-cancer compounds.
Our initial study was carried out both in solvent-free conditions as well as under various solvents like ethanol, acetonitrile, ethylene dichloride, tetrahydrofuran, dimethylformamide, and water (Table 1).The effect of solvent amount and required time was investigated.Among the mentioned solvents, ethanol gave the best outcomes with an excellent yield.It was found that using more solvent caused the reaction to take 3.5 hours to complete under reux conditions.However, when the minimum amount of ethanol (0.5 ml) was utilized at 100 °C, the reaction mixture solidied within the 35 min but remained slightly incomplete.Hence, we decided to use 0.5 ml more ethanol to the reaction mixture and stir for 25 minutes.This successfully completed the reaction, and we observed a highyield conversion of DHPM.Having set optimized parameters, La(OTf) 3 -catalyzed 20 derivatives of dihydopyrimidones were synthesized by employing various substrates (Table 2).All the synthesized compounds were characterized by 1 H nuclear magnetic resonance (NMR), 13C{ 1 H} 35 attached proton test, 49 and either high-resolution mass spectra (HRMS) or liquid chromatography-mass spectrometry (LCMS) analysis (see ESI †).
Aer improving the reaction, the substrate scope for the La(OTf) 3 -catalyzed synthesis of dihydropymidine derivatives was investigated by changing the different acetoacetanilide derivatives.Substitution of the acetoacetanilides gave a slight variation in the time of reaction and yield of the product (Table 2).Compound 4a was obtained with an excellent 97% yield in one hour (Table 2, entry 1), while when 3,4-(CF 3 ) 2 substituted acetoacetanilide derivative was employed under the same reaction condition, a slightly decreased yield of 79% was observed (Table 2, entry 11) in 1.5 h.When chloro substituted acetoacetanilide derivatives were used as a substrate, a comparatively good yield was observed than the methylsubstituted acetoacetanilide derivatives (Table 2).In the case of triuoro methyl substituted acetoacetanilide derivative, it takes 1.5 h to complete the reaction.Based on the abovediscussed facts, it was observed that substitutions on the acetoacetanilide have shown great inuence on the reaction time as well as in isolated yield (Scheme 2).
The Biginelli reaction is an example of an acid-catalysed three-component reaction.In the rst step of the mechanism, the acid protonates the aldehyde.The nucleophilic NH 2 group from urea then attacks the electrophilic aldehyde, leading to the formation of an N-acyliminium ion intermediate with the release of a water molecule.Next, the enol form of the b-keto amide attacks the N-acyliminium ion, resulting in the formation of a ureide intermediate.This intermediate subsequently converts into the nal product (Scheme 3).

Biological evaluation
2.2.1 Establishing a potential relationship between structure and biological activity of dihydropymidine derivatives.To determine the biological relevance of these molecules in vitro, a cell viability assay was conducted on all synthesised compounds in triplicates at 25 mM against human cell line U87 MG.Out of the 20 molecules tested, 4f had the maximum potency (Fig. 2A).MDA-MB-231, A549, and HEPG2 cell lines, as well as primary patient derived glioblastoma cells GBM6 and GBM22, were used to assess the anti-cancer properties of 4f in vitro using cell viability assays.Aer 72 h incubation, cell viability was measured, and the EC 50 values for 4f in MDA-MB-   2B).Since 4f treatment leads to reduced cell numbers over 48-96 hours, we wanted to query whether 4f function is cytotoxic or cytostatic.Hence, we carried out an assay to observe induction of apoptosis.We treated GBM6 cells with 25 mM of 4f and utilised FDA-approved drug crenolanib as a positive control.10 mM of crenolanib induced signicant apoptosis in GBM6 cells, however, 4f did not induce apoptotic cell death suggesting that 4f may be cytostatic in nature (ESI Fig. S1 †).2.2.2 4f causes signicant cell cycle delay in cancer cells.Since 4f did not induce apoptosis in cancer cells, we enquired if the cytostatic function observed of 4f could be due to the induction of cell cycle defects.For this purpose, we utilised asynchronous GBM6 cells and treated then with or without 15 or 25 mM 4f over 16 hours.The cells were harvested posttreatment, xed with ice-cold 70% ethanol, and the DNA content were labelled with propidium iodide.Following this, cell cycle stages of the cells were analysed using ow cytometry.Interestingly, 4f treatment led to a dose-dependent increase in G1 and G2/M population of cells with a signicant decrease in Sphase cells (Fig. 3).This clearly suggests that the cytostatic function of 4f is indeed driven by induction of cell cycle delays in cancer cells.
2.2.3 Combination with clinically relevant brain-penetrant drugs.Since 4f induces cytostatic effects through cell cycle impediment, we wanted to test if a combination with clinical drugs could promote synergism in inducing cancer cell death.For this purpose, we tested 4f in two different cell lines (U87 MG and GBM6) in combination with PI-103, buparlisib, abemaciclib, bozitinib, marizomib, nilotinib, and osimertinib (Fig. 4A-G).Interestingly, 4f induced potent cytotoxicity in combination with nilotinib, and osimertinib (Fig. 4F and G) in the cells.While an additive combination effect was observed across all drugs with 4f, the synergistic effect seen in combination with EGFR inhibitor osimertinib was very robust and observed across both cell types.

2.2.4
Establishing cytotoxicity in glioblastoma stem 3D neurospheres.To further determine whether 4f treatment could impact a 3D glioma stem cell culture system, we treated GBM120 glioma stem cell lines with 15 and 25 mM 4f for 13 days.4f treated GBM120 neurospheres were signicantly smaller in size compared to DMSO control as observed under bright eld (Fig. 5), and neurosphere formation was drastically reduced at the highest concentration (Fig. 5).

2.2.5
In silico ADMET prediction.The most crucial stage in the process of discovering and developing new drugs involves anticipating the ADME (Absorption, Distribution, Metabolism, and Excretion) properties.In silico ADMET prediction of most bioactive compound 4f has been performed and their druglikeness has been assessed through ADME prediction.The most potent compound and their prediction of drug-likeness are mentioned in Table 3.The physicochemical property value of 4f lies in the zone of various lters (Table 3).The molecule 4f shows good drug likeness properties as it follows Lipinski, veber, egan and muegge rule.There are two violations in terms of molecular weight and molar refractivity observed in the Ghose rule.This, 4f has drug-likeness with the bioavailability score of 0.55.This drug likeness data of 4f makes it a good drug candidate.Furthermore, other parameters like molecular weight (MW), topological surface area (TPSA), molar refractivity (MR), rotational bond (RB), hydrogen bond donor and acceptors lay in the acceptable range.Other than that, important physicochemical data is indicated in Table 3.The diameter of the neurospheres were quantified using ImageJ.The significance of the differences was measured using one-way ANOVA with Tukey's multiple comparisons for n = 3 biological replicates; ****p < 0.0001.

Conclusion
A series of benzyloxy derivatives of DHPM was synthesised using a multicomponent reaction.Lanthanum triate catalyzed reaction gave good to excellent yield within 1-1.5 h.The advantages of this protocol include good to high yields, operational simplicity, simple ltration and needing no extraction or separation by column chromatography is necessary.Out of the series we identied 4f with the most potent anti-cancer activity in a diverse set of cancer cell lines including primary patientderived cells.Furthermore, in combination with brainpenetrant small molecule kinase and proteasome inhibitors, 4f induced potent cytostatic activity and cell cycle impediment which attests for further medicinal chemistry and development of the benzyloxy derivatives of DHPM backbone.Interestingly, treating patient derived glioma stem 3D neurospheres with 4f signicantly reduced neurosphere diameter (Fig. 5), this suggesting potent anti-glioma activity in stem-like organoid models.Furthermore, 4f exhibited a remarkable potency in combination with EGFR-mutant targeting osimertinib.Although kinase inhibitory activity (ESI Fig. S2 †) or apoptosis (ESI Fig. S1 †) were not observed for 4f, the data do suggest that a future in vivo bioavailable derivative of 4f could be used as a combination with osimertinib to target cancers with mutated/ amplied EGFR.Both osimertinib and marizomib are covalent inhibitors and represent a paradigm shi toward cancer therapeutics.The ability of our drug to efficiently combine with covalent inhibitor provides an impetus toward further SAR and congener exploration to improve the DHPM backbone for clinical readiness.Compound 4f shows the most promising drug candidate due to its favorable drug-likeness and in silico ADMET properties.Therefore, this study establishes a novel synthetic scheme which will allow for the development of future clinically relevant anti-cancer molecules which can be used in combination with specic drugs targeting EGFR mutated cancers.

General
All chemicals were purchased from commercially available sources and used without further purication.Melting points were determined by the open capillary tube method and are uncorrected. 1H NMR and 13 C{1H} NMR, HSQC, and HMBC spectral analysis were recorded on BRUKER AVANCE II 600 NMR Spectrometer equipped with cryogenic TCI probe using DMSO-d 6 as the solvents.Abbreviation used for NMR signal: s = Singlet, d = Doublet, t = triplet, dd = double doublet m = Multiplet.The chemical shis are expressed in parts per million and coupling constants (J) are provided in Hertz.

General procedure for the synthesis of spiroxindoles 4(a-t)
A mixture of 1 mmol 4-((1-benzyl-1H-1,2,3-triazole-4-yl)methoxy) benzaldehyde 1(a-c), 1.1 mmol of acetoacetenilide derivatives 2(a-k), and 1.2 mmol urea derivative 3(a-b) was added to a 50 ml round bottom ask with 0.5 ml ethanol and 0.1 mmol (10 mol%) lanthanum triate.It was stirred at 100 °C for 35 min.Within this time, the reaction mixture solidied or became sticky, and then 0.5 ml more ethanol was added and stirred for the required time, as mentioned in Table 2. Next, 5 ml more ethanol was added and cooled to room temperature.The progress of the reaction was monitored by TLC.Aer completion of the reaction, the reaction mixture was poured slowly into 20 ml ice-cold water with stirring and kept still until the precipitation of the product was completed.The crude product was ltered and washed with 20% aqueous solution of ethanol (5 ml × 3).Products were recrystallized from 6 ml ethanol.
4.3.3Cell viability and apoptosis assays.To measure cell viability, actively proliferating cells were seeded at with an equal number of cells per well.Cell viability assays were carried out with 48-96 h treatment of indicated drugs or DMSO control using CellTiter 96® AQueous Non-Radioactive Cell proliferation assay, adhering to manufacturer instructions.Absorbance was measured using a Tecan multi-well plate reader and data was represented as % viability compared to DMSO treated control as stated previously. 20,51,52For the assessment of apoptosis, Annexin V-FITC (ab176749; Abcam, Cambridge, UK) was used as stated previously. 5316 h post drug treatments, the wells were washed and buffer solution containing Annexin V-FITC diluted as par manufacturer's instructions were added to the wells.The cells were incubated for 90 min prior to imaging.Bright eld and uorescent imaging were taken using a Thermo Scientic EVOS imaging system.
4.3.4Cell cycle analyses.Cell cycle analyses using propidium iodide and ow cytometry were carried out as described previously. 52,54Asynchronous GBM6 cells were treated with either DMSO or 4f at 15 or 25 mM for 16 h.Post treatment, cells were washed with PBS + 1% FBS and resuspended in ow cytometry tubes.Cells were then xed with 70% ice-cold ethanol and propidium iodide (50 mg ml −1 ) was added to the cells and incubated in the dark at room temperature (25 °C) for 30 min.The cell populations were then subjected to quantitative measurement of DNA content by ow cytometry using a FACS-Fortessa (BD Biosciences) and cell cycle distribution and the percentage of G 2 /M-S-G 1 cells to the total cell events were determined by the BD FACS Diva soware.Stacked bar graphs were derived using Graphpad Prism.
4.3.5 Neurosphere formation assay.Neurosphere formation assay was carried out as stated previously. 20Briey, GBM120 cells were plated at 4000 cells per well in neurosphere media supplemented with either DMSO, 15 mM and 25 mM of 4f for 13 days in triplicates.Aer 13 days, representative images were taken of each well using the Zeiss Axiovert Live microscope.Diameters of the neurospheres were quantied using ImageJ soware, and graphs were plotted on GraphPad Prism.
4.3.6Kinase screen analysis of 4f.Kinase inhibitor speci-city proling assays were carried out at The International Centre for Protein Kinase Proling (http://www.kinasescreen.mrc.ac.uk/) as stated previously. 20Briey, 4f biochemical kinase inhibitory property was determined against a panel of 139 protein kinases as described previously. 55,56Protein kinases were assayed in vitro with 10 mM nal concentration of 4f and results are presented as a percentage of kinase activity to DMSO control reactions as an average of triplicate reactions in the form of comparative histograms using Adobe Illustrator.
4.3.7 Statistical analysis.All analysis was conducted using Graphpad Prism statistical package and presented as mean ± SD unless otherwise stated.

Fig. 2 4f
Fig. 2 4f exhibits anti-cancer activity in diverse cancer cell lines.(A) Cell viability of a panel of DHPM derivatives 4a-t at 25 mM against human U87 cell line was assessed after 96 hours using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit.Viability of DMSO-treated cells was used as a control.Data are represented as fold viability of DMSO-treated control.(B) Indicated cell lines were treated with various doses of 4f for 72 hours and their viability was measured using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit.Viability of DMSO-treated cells was used as control.Data are represented as fold viability of DMSO-treated control for each cell line with n = 3 biological replicates.See also ESI Fig. S1.†

Fig. 4
Fig. 4 4f induces enhanced cytotoxicity in combination with specific clinically relevant brain-penetrant small molecule inhibitors.U87 and GBM6 cells were treated with 4f alone (10 and 25 mM respectively) or clinically relevant brain-penetrant drugs alone (A) PI-103 100 nM, (B) buparlisib 500 nM, (C) abemaciclib 1 mM, (D) bozitinib 7 mM, (E) marizomib 400 nM, (F) nilotinib 5 mM, (G) osimertinib 2 mM, or a combination C of both for 48 hours and cell viability was analysed by CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit.Viability of DMSO-treated cells was used as control.Data are represented as fold viability of DMSO-treated control for each cell line (* indicates statistical significance compared to each single compound treatment; one-way ANOVA with Dunnett's multiple comparison; ns: not significant).Same dataset for 4f was used across all combination studies from (A)-(G).(H) Table lists the cellular targets of the respective clinically relevant brain-penetrant drugs.

Fig. 5
Fig. 5 Compound 4f induces anti-glioma activity in 3D glioblastoma cell cultures.GBM120 cancer stem cells were treated with either DMSO or 15 mM or 25 mM or 4f for 13 days and neurospheres were allowed to form.A representative image of neurospheres is shown.Scale bar = 125 mm.The diameter of the neurospheres were quantified using ImageJ.The significance of the differences was measured using one-way ANOVA with Tukey's multiple comparisons for n = 3 biological replicates; ****p < 0.0001.

Table 1
Effect of solvent on the reaction a More than 0.5 ml solvent.d 0.5 ml solvent.
bObservation from TLC analysis.c